3.3 Screening of conjugates in cell proliferation activity and
scratch wound healing assays
The efficacy of FGF2 conjugates for wound healing can be assessed by
measuring the cell proliferation, migration, and vasculogenesis of
dermal fibroblasts and endothelial cells (Rabuka, 2010; van Horssenet al. , 2006). Here, human dermal fibroblast cells (HDFs) and
human umbilical vein endothelial cells (HUVECs) were used as in
vitro models to screen and compare the bioactivities of these
conjugates.
First, the dose-dependent effects of
PEG-FGF2 conjugates on cell proliferation of HUVECs were investigated by
CCK-8 assay. The cells were incubated with various treatment conditions
at concentrations of 0.1, 1, and 10 ng·mL-1 of protein
for 24 h. Compound 6 promoted significantly greater cell
proliferation than all the other compounds, including native FGF2, even
at a low concentration (0.1 ng·mL-1), with the highest
pro-proliferation activity at a concentration of 10
ng·mL-1 (Figure
3A ). We also evaluated the cell
proliferation activity of these conjugates by immunofluorescence
staining for Ki67-positive HUVECs at 10 ng·mL-1.
Compounds 5 and 6 showed a significant increase in the
number of Ki67-positive cells compared with the PBS control, with a
bigger effect for compound 6 than that for FGF2 (Figure
3 B-C ).
Next, a scratch wound healing assay was performed to evaluate the
effects of PEG-FGF2 conjugates on cell migration of HUVECs. A scratch
was made through a confluent layer of growth factor starved cells
followed by the application of different compounds. Compared with FGF2
treatment, a significant reduction in wound area was observed upon
treatment with compound 6 at 24 h, with migration of HUVECs
increased by ~163% compared to only
~108% for FGF2 (Figure 3 D-E ). The activity of
compound 5 is similar to that of FGF2. Overall, these results
indicate that compound 6 can notably promote proliferation and
migration of HUVECs relative to unmodified FGF2.
The same compounds were also tested using human dermal fibroblasts
(HDFs). There was increased proliferation and migration of HDFs after
treatment with compound 6 relative to treatment with other
protein conjugates (Figure 4 A-C ). These results were
consistent with the results for HUVECs, which indicated improved
bioactivity of compound 6 to promote cell proliferation and
scratch wound healing in both dermal fibroblasts and endothelial cells,
which may be attributed to the long-acting effect of PEGylation.